1. Maintenance and expression of genetic information Central Dogma: DNA RNA Protein

3. GAATTGCGCCTTTTG

4. 5’-GAATTGCGCCTTTTG-3 3’-CTTAACGCGGAAAAC-5’

8. Minor Groove Major Groove

11. DNA can be supercoiled

12. Semi-conservative Replication of DNA The Watson-Crick Model

13. Proof of the Watson-Crick Model: The Meselson-Stahl Experiment

14. # generations 0 0.3 0.7 1 1.1 1.5 1.9 2.3 3 4.1 0 and 1.9 mixed 0 and 4.1 mixed

15. Starting DNA Heavy/Heavy 1st generation All Heavy/Light 2nd generation Two Heavy/Light Two Light/Light 3rd generation Two Heavy/Light Six Light/Light The Meselson-Stahl Experiment

17. DNA Polymerase

18. A 3’ hydroxyl group is necessary for addition of nucleotides

19. 1’ 2’ 3’ 1’ 2’ 3’ 4’ 5’ 4’ 5’ 2’ 3’ 2’ 3’ 4’ 5’ 4’ 5’ 1’ DNA chain growth is driven by PPi release/hydrolysis

20. Accuracy of DNA polymerases is essential. --Error rate is less than 1 in 10 8 --Due in part to “reading” of complementary bases --also contains its own proofreading activity

21. DNA Polymerase contains a Proofreading subunit

22. Proofreading by DNA polymerase

23. Proofreading by DNA polymerase

25. Both Template strands are copied at a Replication Fork

26. The polarity of DNA synthesis creates an asymmetry between the leading strand and the lagging strand at the replication fork

27. Topoisomerase Protein complexes of the replication fork

28. Protein complexes of the replication fork: DNA polymerase DNA primase DNA Helicase ssDNA binding protein Sliding Clamp Clamp Loader DNA Ligase DNA Topoisomerase

29. DNA primase synthesizes an RNA primer to initiate DNA synthesis on the lagging strand

30. Replication of the Lagging Strand

31. DNA ligase seals nicks left by lagging strand replication

32. DNA helicase unwinds the DNA duplex ahead of DNA polymerase creating single stranded DNA that can be used as a template

33. DNA helicase moves along one strand of the DNA

34. ssDNA binding proteins are required to “iron out” the unwound DNA

35. ssDNA binding proteins bind to the sugar phosphate backbone leaving the bases exposed for DNA polymerase

36. DNA polymerase is not very processive (ie it falls off the DNA easily). A “sliding clamp” is required to keep DNA polymerase on and allow duplication of long stretches of DNA

37. A “clamp loader:” complex is required to get the clamp onto the DNA

38. Lagging strand synthesis

39. MCM proteins PCNA RPC Topoisomerase

40. Ahead of the replication fork the DNA becomes supercoiled

41. The supercoiling ahead of the fork needs to be relieved or tension would build up (like coiling as spring) and block fork progression.

42. Supercoiling is relieved by the action of Topoisomerases. Type I topoisomerases: Make nicks in one DNA strands Can relieve supercoiling Type II topoisomersases Make nicks in both DNA strands (double strand break) Can relieve supercoiling and untangle linked DNA helices Both types of enzyme form covalent intermediates with the DNA

43. Topoisomerase I Action

44. Topoisomerase I Action

45. Topoisomerase II Action

46. Topoisomerase II Action

47. Topoisomerases as drug targets: Because dividing cells require greater topoisomerase activity due to increased DNA synthesis, topoisomerase inhibitors are used as chemotherapeutic agents. E.g. Camptothecin -- Topo I inhibitor Doxorubicin -- Topo II inhibitor These drugs act by stablilzing the DNA-Topoisomerase complex. Also, some antibiotics are inhibitors of the bacterial-specific toposisomerase DNA gyrase e.g. ciprofloxacin

48. DNA is replicated during S phase of the Cell Cycle

49. In S phase, DNA replication begins at origins of replication that are spread out across the chromosome

50. Each origin of replicaton Initiates the formation of bidirectional replication forks

51. Origins or replication are strictly controlled so that they “fire” only once per cell cycle Errors lead to overreplication of specific chromosomal regions. (= gene amplification) This seen commonly in cancer cells and can be an important prognostic indicator. It can also contribute to acquired drug resistance. E.g. Methotrexate induces amplification of the Dihydrofolate Reductase locus.

52. Errors of DNA Replication and Disease The rate of misincorporation of bases by DNA polymerase is extremely low, however repeated sequences can cause problems. In particular, trinucleotide repeats cause difficulties which can lead to expansion of these sequences. Depending where the repeat is located expansion of the sequence can have severe effects on the expression of a gene or the function of a protein.

53. Several mechanisms for the expansion of trinucleotide repeats have been proposed, but the precise mechanism is unknown. From Stryer: Looping out of repeats before replication.

54. Several inherited diseases are associated with expansion of trinucleotide repeat sequences. Very different disorders, but they share the characteristic of becoming more severe in succeeding generations due to progressive expansion of the repeats